Confocal microscopy was used to investigate the effects of the PI 3-kinase inhibitor wortmannin in isolated rat adipose cells. In combination with previous kinetic biochemical studies, the results suggest that: (i) insulin stimulates the exocytosis of GLUT4 glucose transporters through a direct pathway from a specialized basal compartment to the plasma membrane, (ii) during endocytosis in the presence of insulin, GLUT4 is sorted out of the transferrin receptor compartment into a separate recycling pathway back to the plasma membrane, and (iii) both of these pathways involve wortmannin-sensitive enzymes. To study the role of the GTPase dynamin in GLUT4 intracellular recycling, we have overexpressed dynamin-1 wild type and a GTPase-negative mutant (K44A) in primary rat adipose cells. The data demonstrate that the endocytosis of GLUT4 is largely mediated by processes which require dynamin. To study the role of tyrosine- phosphorylated proteins such as insulin receptor substrate (IRS- 1, IRS-2, Grb2-associated binder-1, and pp60 (IRS-3) in glucose transport, we analyzed adipose cells from IRS-1 null mice. The data suggest that both IRS-1 and pp60 play a major role in insulin-induced glucose transport in adipose cells, and that pp60 is predominantly involved in regulating this process in the absence of IRS-1. To test the hypothesis that glucocorticoids reduce insulin- stimulated skeletal muscle glucose transport by inhibiting the recruitment of GLUT4 to the cell surface, we determined the effect of glucocorticoid treatment on cell-surface GLUT4. The findings indicate that glucocorticoid-induced inhibition of insulin-stimulated glucose transport in muscle is due to impaired GLUT4 translocation. To determine which GLUT isoform is responsible for the noradrenaline-induced increase in glucose transport into intact brown adipocytes in culture, we labeled the exofacial glucose- binding sites of GLUT1 and GLUT4 with a membrane-impermeant photoaffinity reagent, ATB-[3H]BMPA. The results demonstrate that noradrenaline stimulates glucose transport in brown adipocytes by enhancing the functional activity of GLUT1 through a cAMP-dependent mechanism. In order to evaluate the presumed importance of GLUT2 in maintaining the efficiency of beta-cell glucose uptake, we studied the kinetic characteristics of 3-0-methyglucose uptake in two beta-cell lines; one of these is the beta TC3 cell line which expresses GLUT1 and the other is the beta HC9 cell line which expresses both GLUT1 and GLUT2. Our results suggest that GLUT1 is able to compensate for GLUT2 loss as it occurs in beta TC3 cells and maintains an high enough capacity of glucose uptake to sustain glucose metabolism in pancreatic beta-cells.